Within this application several publications are referenced by number within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Acquired Immune Deficiency Syndrome (AIDS) is a new epidemic characterized by a marked depletion of the cellular immune response. The causative agent of the disease is now firmly established to be the human retrovirus known as Human Immunodeficiency Virus Type 1 (HIV-1), but was formerly known as Human T-cell lymphotropic virus III or Lymphadenopathy virus (HTLV-III/LAV) (1-4). Efforts to arrest the spread of this virus are being made on two broad fronts: the development of antiviral vaccines which might allow immunized individuals to resist infection, and the development of antiviral drugs which would specifically retard or arrest viral replication. One potentially important target of such drugs is the virion-associated enzyme reverse transcriptase (5-8).
In the early stages of the retroviral life cycle, viral RNA is copied to form a double-stranded DNA, which is integrated into host DNA to generate the provirus (for review, 1). The synthesis of the proviral DNA is catalyzed by the enzyme reverse transcriptase, which may efficiently utilize either RNA or DNA templates for DNA synthesis by the elongation of a primer bearing a paired 3' hydroxyl terminus. Inherent in the same protein is a second activity, RNAse H, which degrades RNA present as a duplex RNA:DNA hybrid. The viral pol gene encodes many enzymatic activities which participate in various steps of the life cycle. The pol gene product is initially expressed as a polyprotein Pr200gag-pol (2,3), containing sequences encoded by the gag gene fused to sequences encoded by the pol gene; proteolytic processing is required to remove the sequences and to excise the mature products from the pol sequences.
Reverse transcriptase is widely used as a means of producing complementary DNA (cDNA) copies of messenger RNA (mRNA) molecules. These cDNAs may be inserted into expression vectors which are used to transform cells so that the resulting cells produce a desired polypeptide encoded by the original mRNA.
The HTLV-III reverse transcriptase has been purified in small quantities and some of its properties are established. The enzyme has unusual template and divalent cation preferences (9-11), suggesting that specific inhibitors of the enzyme may be discovered. Certain inhibitors of HTLV-III reverse transcriptase activity may be used to block the activity of this enzyme as it is produced by viruses infecting human cells and tissues.
Reverse transcriptase being an integral part of the viral life cycle, this will allow use of the inhibitors to treat or prevent HTLV-III related diseases. The isolation of such inhibitors would be facilitated by the availability of larger quantities of the active enzyme.
Reverse transcriptase produced by and isolated from virions is commercially available. However, it is quite expensive due to the low abundance of the gene product in the virions.
Several disclosures in the art concern the production of a polypeptide having reverse transcriptase activity by bacteria transformed with genetically engineered vectors. One disclosure involves the shotgun cloning into Escherichia coli of total genomic DNA isolated from the cells of warm-blooded vertebrate animals, e.g. fowl liver cells, [Japanese patent publication no. 56087600].
Two other disclosures detail the expression of a segment of the retrovirus pol gene to produce reverse transcriptase in E. coli in high yield (16, 17).
Co-pending, co-assigned U.S. patent application Ser. No. 731,128, filed May 6, 1985 describes the expression of enzymatically active reverse transcriptase from Moloney murine leukemia virus (MuLV) in relatively high yield.
The present invention uses a modified region of the HTLV-III pol gene which is inserted into a plasmid, its transcription being controlled by an inducible promoter. The expression of the inserted gene fragment results in the production of a polypeptide with HTLV-III reverse transcriptase activity.
Additionally, the present invention describes a method for identifying substances which inhibit HTLV-III reverse transcriptase activity.